The goal of this research is to elucidate the fundamental mechanisms by which Na+ transport influences Ca2+ homeostasis and signaling, in neurons and glia. Neurons and glia both express Na+ pumps with the a1 isoform of the catalytic (a) subunit and Na+ pumps with either the ot2 (glia) or a3 (neurons) isoform. Na+ pumps with a2 or a3 isoforms are localized to plasma membrane-endoplasmic reticulum (PM-ER) junctional complexes ("PLasmERosomes") and are coupled to PM Na/Ca exchangers (NCX) and, thus, to cytosolic ([Ca2+]CYr) and ER Ca2+ concentration and Ca2+ signal regulation in these cells. Critical questions are: How are the a2 and oc3 Na+ pumps sorted and tethered to their appropriate PM destinations? And, what are the local and global functional consequences of this special organization? There are four Specific Aims: Aim 1. To determine how Na+ pump a2 and a3 subunits are targeted and tethered to their appropriate PM locations, a subunit chimeras (e.g., part a2 and part a1, and vice-versa), WT and mutated a truncations, and ankyrin B knockout mice will be used to test the hypothesis that ot2and a3 subunits are targeted to PLasmERosomes by specific N-terminal amino acid (AA) sequences and are tethered by ankyrin B. Aim 2. To determine whether the sub-PM Ca2+ concentration at PM-ER junctions is controlled independently of "bulk" [Ca2+]CYT- Novel near-membrane Ca2+ indicators (FFP-18 and G-CaMP-2, an engineered protein targeted to the PM- ER junction) will be used to test, directly, the idea that PM-ER junctional space Ca2+ is regulated by oc2/a3 Na+ pumps and NCX1 in astrocytes and neurons. Aim 3. To determine how linkage of the Na+ pump a2 subunit isoform, NCX1, and ankyrin B contributes to their central roles in local Ca2+ regulation and global Ca2+ signaling in astrocytes. Aim 4. To determine the roles of the Na+ pump and NCX in Ca2+ efflux from neuronal dendrites and nerve terminals and how these processes influence neuronal function. For Aims 3 and 4, null mutant mice, and molecular biological and pharmacological tools will be used to test the hypothesis that structural linkage of key PM Na+ and Ca2+ transporters in PLasmERosomes enables them to serve critical functionally-coupled roles in Ca2+ regulation and signaling in neurons (Aim 4) and in astrocytes (Aim 3). These studies will shed new light on specific mechanisms that regulate normal Ca2+ homeostasis and signaling, and that may go awry during hypoxia/ischemia and other brain pathologies.